In ovo protection against infectious bronchitis

ABSTRACT

The present invention is directed to processes and compositions for protecting host animals (e.g., chickens) from exposure to virulent infectious bronchitis virus. In ovo administration of live, avirulent strains of IB at appropriate dosage levels on a per egg basis provides an effective and efficient vaccination having acceptable safety and efficacy features.

[0001] This application claims the benefit under 35 U.S.C. §119(e) toU.S. application Ser. No. 09/775,750, filed Feb. 2, 2001, which in turnis a non-provisional filing of application No. 60/185,631 filed on Feb.29, 2000, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention is directed to novel ways of providing in ovoprotection against infectious bronchitis (hereinafter, “IB”) in hostanimals such as chickens. More particularly, vaccines derived fromtraditional commercially-available IB vaccines have proven to be bothsafe and efficacious upon appropriate in ovo administration to hostanimals as described herein.

BACKGROUND OF THE INVENTION

[0003] IB is a highly infectious/transmissible respiratory disease thataffects chickens of all ages. The disease of IB is caused by a virus ofthe coronavirus group. IB disease symptoms vary widely; however,reported effects include death, respiratory tract distress, depressedproduction, decreased peak production of eggs, abnormalities in theeggshells, diarrhea, and a nephrosis/nephritis syndrome. Undesiredweight loss in young chicks and/or insufficient/low-quality productionof eggs from laying flocks are commercially-significant adverse impactsof IB disease in chickens.

[0004] Commercially-available vaccines for IB are not administered inovo. Rather, they are administered post-hatch in a variety of formats.Briefly, such vaccines are typically administered by the labor-intensivemethods of spraying (e.g., hand spray, knapsack spray, or automatedspray equipment) or in drops (eye or nose).

[0005] As more fully explained below, the in ovo vaccines of the presentinvention provide distinctive advantages over the inconvenient andtime-consuming post-hatch routes of administration presently available.

SUMMARY OF THE INVENTION

[0006] In brief, the present invention is directed to in ovo vaccines(hereinafter, “IOV”) derived from commercially-available IB vaccines.Experimental results establish the safety and efficacy of the IOVrelative to in ovo administration to chickens. Appropriate dosingparameters have also been developed. In addition to the IOV, theinvention contemplates related compositions suitable for in ovoadministration.

[0007] Processes for protecting a host animal against IB are also withinthe ambit of the present invention. Such administration provideseconomic advantages in that in ovo vaccination is easier, and faster andutilizes a smaller dose of vaccine than conventionally administeredvaccines.

DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention is directed to in ovo inoculations ofanimals, in particular, poultry. As that term is used herein, poultryrefers to any bird or fowl which is bred commercially, and thereforeincludes chickens, turkeys, ducks, geese, bantams, quail, pigeons andthe like. Of particular interest are chickens.

[0009] All types of vaccines for inoculation are contemplated for use inthe invention, and in particular, a vaccine against infectiousbronchitis, or IB. The vaccines may be obtained from whatever source isavailable in the industry. A preferred vaccine is an IB vaccine marketedunder the trademark POULVAC®, which is available from Fort Dodge AnimalHealth in Fort Dodge, Iowa or Weesp, the Netherlands. Other IB vaccinesmay also be utilized. The vaccines of the invention preferably containlive, nonvirulent strains of the infectious agent, including IB. Thevaccines should induce an immunogenic response in the host, generatingthe production of antibodies sufficient to confer immunity.

[0010] The quantity of pathogenic agent to be included in the vaccine tobe administered to the host egg can vary, depending upon the particularpathogenic agent, and also the size of the animal (larger animals mayrequire larger quantities of agent). A desired quantity is within therange of about 10^(−1.0) EID₅₀ to about 10^(2.0) EID₅₀ of pathogenicagent, e.g. virus (in particular IB), per vaccine dose. A quantitywithin the range of about 10^(0.0) EID₅₀ to about 10^(1.0) EID₅₀pathogenic agent per vaccine dose is also useful herein. Throughout thisapplication, “EID₅₀” refers to a 50% egg infectious dose.

[0011] In addition to the foregoing, the vaccines may be formulated withknown additives, including adjuvants. Examples of desirable adjuvantsinclude polymers and copolymers of acrylic acid, as well as thosederived from other alkyl esters. Other constituents include media suchas water, saline solution, or water-in-oil emulsions in quantitiessufficient to top off the dose. The pathogenic agent may be dissolved orsuspended in the media just described. In a preferred embodiment of theinvention, the vaccine is formulated with substantially no virusneutralizing factor.

[0012] A vaccine dose is typically within the range of about 0.001 mL toabout 1.0 mL, and more preferably within the range of about 0.01 to 0.1mL, with about 0.05 mL being even more preferred.

[0013] The dosing regimen for the vaccine most desirably includesadministration in ovo to a developing chick in a fertilized egg that hasnot yet hatched. One administration of the dose is typically preferred,but more than one is within the scope of the invention. The dosingschedule chosen should ensure both the safety of the developing animal,as well as efficacy of immunization.

[0014] A dose of vaccine may be administered in ovo during a time periodwhich is within the range of about day 1 up to and including about a fewminutes before hatching. More preferably, a dose is delivered in ovowithin the time period of about day 5 to about day 25. Even moredesirably, the dose is administered during the period of about day 10 toabout day 20. A dosing at about day 18 may be particularly desirable.

[0015] Administration of the vaccine may be done by hand, but is moretypically and economically administered using commercially available egginjection equipment, such as that available from Embrex, Inc. of NorthCarolina.

[0016] An advantage of in ovo vaccine according to the invention is thatthe vaccine is applied to each individual bird (egg). This translatesinto better accuracy as compared with more traditional vaccinationprograms (non-in ovo). This is reflected in the high percentages ofprotection against challenge in the vaccinated birds. In addition,because the in ovo birds are vaccinated at a considerably earlier agethan are those who receive the inoculation post-in ovo, there is moretime for the birds to develop their immunity before exposure to outsideambient conditions in which virulent strains of the virus may bepresent. This result has been unexpected. Normally, introduction of liveviruses into embryos would have been expected to generate fairly lethalresults. A developing embryo is a highly fragile organism, and thepresence of a live virus such as IB would have normally killed thedeveloping animal. At best, much of the art has mandated the use of avirus neutralizing factor to prevent such an occurrence when chicks areinoculated in ovo. Conversely, introduction of what would have beenconsidered exceedingly minute quantities of live virus, while notkilling the embryo, would not have been expected to impart satisfactoryimmunogenic properties to the organism.

[0017] The following examples are provided by way of illustration, andshould not be construed as limiting the scope of the invention.

[0018] A) The Vaccine.

[0019] The IOV were prepared using a commercially-available IB vaccine(Poulvac® IB MM) from Fort Dodge Animal Health in Fort Dodge, Iowa orWeesp, The Netherlands by reconstitution with saline to a concentrationof 10^(2.0), 10^(1.0), 10^(0.0) and 10^(−1.0) of IB/virus vaccine perdose (0.05 ml). Poulvac® IB MM contains infectious bronchitis virusstrain 1263 of the Massachusetts serotype. This commercially-availableIB vaccine has not been approved or indicated for in ovo administration.

B) EXAMPLE 1 Safety Study for in Ovo Chicken Vaccination

[0020] Specific-pathogen-free (hereinafter, “SPF”) chicken eggs werecommercially obtained from Charles River SPAFAS, Inc. [190 Route 165,Preston, Conn. 06365]. In brief, SPF eggs from SPAFAS were incubatedwithin appropriate facilities. At 18 days of incubation, 4 groups of 25eggs were administered in ovo vaccinations using graded doses of the IOVderived from the Poulvac® IB MM.

[0021] The IOV of the present invention were prepared as follows. Acommercially-available IB vaccine (Poulvac® IB MM) from Fort DodgeAnimal Health in Fort Dodge, Iowa or Weesp, The Netherlands wasobtained. This vaccine contains live, attenuated IB virus in afreeze-dried environment. Prior to its use herein, this vaccinecontained a titer of 10^(6.4) EID₅₀ IB virus per vial. Next, thisvaccine was reconstituted in saline and then further admixed with salineuntil the following concentrations were obtained: solutions containing,respectively, titers of 10^(2.0), 10^(1.0), 10^(0.0), and 10^(−1.0) ofIB virus/vaccine per dose (size: 0.05 ml) were prepared.

[0022] At 18 days of incubation, Groups 1-4 (each consisting of 25 eggs)were injected in ovo with a dose of 0.05 ml per egg of the vaccines ofthe present invention containing, respectively, the following titers ofIB virus: titers of 10^(2.0), 10^(1.0), 10^(0.0), and 10^(−1.0) of IBvirus/vaccine per dose. As a control, Group 5 (also consisting of 25eggs) did not receive any in ovo injections at day 18 of incubation. Toadminister the injections, commercially-available equipment (Inovoject®egg injection machine) from Embrex, Inc. [P.O.B. 13989, ResearchTriangle Park, North Carolina 27709-3989] was used according tomanufacturer's instructions.

[0023] Until hatching, both the inoculated eggs (i.e., 100 eggs total inGroups 1-4 @25 eggs/group) and the control eggs (i.e., 25 eggs in Group5) were incubated within the same incubator. The number of hatched eggsper group was experimentally recorded at days 20, 21, and 22 ofincubation.

[0024] Tables 1 and 2 presents the results obtained. TABLE 1 CalculatedHatchability Results Hatchability results after in ovo vaccination atincubationday 18 with a dose of 0.05 ml/egg of IB vaccine to Groups 1-5(25 eggs/group) Group EID₅₀ # hatched % hatched 1 10^(2.0) 17 72 210^(1.0) 21 84 3 10^(0.0) 17 72 4  10^(−1.0) 20 80 5 None 24 96

[0025] TABLE 2 Raw Hatchability Results Hatchability results after inovo vaccination at incubation day 18 with a dose of 0.05 ml/egg of IBvaccine to Groups 1-5 (25 eggs/group) # hatched # hatched # hatchedtotal # Group EID₅₀ (day 20) (day 21) (day 22) hatched 1 10^(2.0) — 12 517 2 10^(1.0) — 10 11  21 3 10^(0.0) — 12 5 17 4  10^(−1.0) — 17 3 20 5none — 24 — 24

[0026] Hatchability in the inoculated eggs ranged from 72% to 84% incomparison to 96% for the negative control eggs of Group 5. All of theseobserved hatchability percentages were within customary limits. Nosystemic effects between the inoculated groups (i.e., Groups 1-4) wasobserved.

[0027] Based upon the results of Experiment 1 as set forth above, it wasconcluded that in ovo vaccination at incubation day 18 using dosagesranging from a low of 10 ^(1.0) EID₅₀ IB vaccine to a high of 10^(2.0)EID₅₀ IB vaccine was safe relative to hatchability.

C) EXAMPLE 2 Efficacy Study for in Ovo Vaccination of SPF Chicken Eggs

[0028] SPF chicken eggs were obtained from Charles River SPAFAS, Inc.All 200 eggs were incubated in appropriate facilities. At 18 daysincubation, all eggs were candled; 14 eggs were unfertilized and within20 eggs the embryos had died. The eggs were divided into 5 groups with25 eggs/group.

[0029] The vaccine to be administered in ovo was prepared in accordancewith the procedures of Example 1 above.

[0030] At 18 days of incubation, similar to the protocol used in Example1 above, Groups 1-4 were injected in ovo with the following titers ofEID₅₀ of IB vaccine/virus in a dose of 0.05 ml/egg: 10^(2.0), 10^(1.0),10^(0.0), and 10^(−1.0). As a control, Group 5 eggs were not injectedwith any vaccines at incubation day 18.

[0031] Next, the inoculated and the control eggs were placed in separateincubators (without turning) for each group of eggs and were left tohatch in the isolation pen in which they were housed. The number of eggshatched was experimentally recorded at days 20, 21, and 22 ofincubation. At 22 days of incubation, all remaining eggs were removedfrom the incubators.

[0032] The chicks were housed in their respective isolation pens withpositive air pressure. All chicks were kept on wood shavings. The roomswere provided with heater lamps to create local temperaturessubstantially above the room temperatures. Chicks were able to choosetheir preferred temperature by adjusting their distance from the heatinglamp. All chicks were fed ad lib and drinking water was ad lib availablein automatic drinkers. Within a week after hatching, all chicks weretagged with an identifying wing mark that contained both a color and anumber. Chicks to be challenged (as described below) at 4 weeks of agewere moved to 1 animal room operating with positive air pressure justbefore the challenge. Additionally, the chicks were experimentallyobserved for clinical signs of IB throughout the study.

[0033] Tables 3 and 4 present the hatchability results. TABLE 3Calculated Hatchability Results hatchability results after in ovovaccination at incubation day 18 with a dose of 0.05 ml/egg of IBvaccine to Groups 1-5 (25 eggs/group) Group EID₅₀ # hatched % hatched 1 10^(2.0)  8 32 2 101.0 10 40 3  10^(0.0) 11 44 4  10^(−1.0) 14 56 5none 20 80

[0034] TABLE 4 Raw Hatchability Results hatchability results after inovo vaccination at incubation day 18 with a dose of 0.05 ml/egg of IBvaccine to Groups 1-5 (25 eggs/group) # hatched # hatched # hatchedtotal # Group EID₅₀ (day 20) (day 21) (day 22) hatched 1 10^(2.0) — 1 78 2 10^(1.0) — 2 8 10 3 10^(0.0) — 8 3 11 4  10^(−1.0) — 6 8 14 5 none —10 10 20

[0035] The observed hatchability was very low for all inoculated groups(it ranged from 32% to 56%) and decreased with increasing vaccine dose.In the control eggs of Group 5, 80% of the eggs hatched. It wasconcluded that, with respect to hatchability, the results obtained werenot representative and were attributed to poor egg quality rather thanto adverse effects of in ovo vaccinations with IB virus. As described indetail in Example 1, acceptable hatchability results were previouslyobtained.

[0036] An analysis of clinical signs (e.g., mortality) in non-challengedchicks further strengthened the above conclusion that the hatchabilityresults were not representative. Table 5 below presents the mortalitydata for hatchlings/chicks from both the 4 vaccinated groups (i.e.,Groups 1-4) and the 1 negative control group (i.e., Group 5). In Table5, “PH” is an abbreviation for post-hatch. The observed clinical signsincluded the following results. Due to diarrhea, several chicks in allgroups were in bad condition. Some chicks exhibited respiratory problemsor umbilical hernia. A total of 5 chicks died (post-hatch) from yolk sacinflammation as follows: in Group 1, 1 chick died; in Group 2, 3 chicksdied; and in group 4, 1 chick died. Post-hatch mortality results(including the deaths attributed to yolk sac inflammation) are presentedin Table 5. For Group 1, 1 dead chick was not observed (which explainsthe finding at day 21 PH that 4 chicks in Group 1 were alive). In Group5, 30% (i.e., 6 hatchlings/chicks) died shortly after hatching. Atcandling prior to in ovo vaccination, embryos in 10% of the eggs haddied. These pre-inoculation deaths, in combination with the describedmortality features of this study, established that inferior egg quality(rather than adverse effects attributable to the in ovo vaccinations)was responsible for both the hatchability results and the observedclinical signs prior to challenge. TABLE 5 Clinical Signs InNon-Challenged Chicks post-hatch (PH) mortality prior to challenge Group1 Group 2 Group 3 Group 4 Group 5 # days PH (n = 8) (n = 10) (n = 11) (n= 14) (n = 20) 0 1 6 1 1 1 1 3 1 1 4 1 5 1 6 1 # live chicks 4 6 11 1214 (day 21 PH)

[0037] A challenge study was conducted as follows. In brief, virulent IBM41 virus was commercially-obtained from the Poultry Health Institute,Doorn, The Netherlands. This challenge virus had a titer of 10^(5.9)EID₅₀ per vial. A final solution containing 10^(4.5) EID₅₀ per ml ofchallenge virus was prepared by appropriate reconstitution withdemineralized water and dilution with nutrient broth of 1 vial of thechallenge virus.

[0038] At 4 weeks of age, all vaccinated and control chicks werechallenged by administration of 10^(3.5) EID₅₀ in 0.1 ml (0.05 ocularand 0.05 intranasal) virulent IB M41 virus per chick. The chicks wereevaluated using the cilia stopping test (hereinafter, “CST”). In brief,6 days post-challenge (hereinafter, “PC”), the chicks were killed andtheir tracheas removed. One part per trachea was collected formicroscopic examination and cillary activity was assessed. Thisassessment used the following scale: +=full movement; ±=impairedmovement; and −=no movement. In cases of impairment, additional tracheaparts were taken to confirm this finding. The percentage of protectionagainst challenge with this virulent strain of IB was calculated usingthe following formula: protection %=(A+{fraction (1/2B)})(100)/C whereinA=# chicks assessed+; B=# chicks assessed±; and C=total # chicks.

[0039] Tables 6 and 7 presents the results of the challenge study. TABLE6 Calculated Results Of Post-Challenge Protection Protection againstchallenge at 3 weeks of age with virulent IB virus as determined by CSTGroup (EID₅₀) # chicks protection % 1 (10^(2.0)) 4 100 2 (10^(1.0)) 6 923 (10^(0.0)) 11 100 4 (10^(−1.0)) 12 100 5 (none) 14 0

[0040] TABLE 7 Raw Results Of Post-Challenge Protection Post- challengeassessments of protection with CST techniques Group +(full CST±(impaired −(no CST (EID₅₀) # chicks motion) CST motion) motion 1(10^(2.0)) 4 4 2 (10^(1.0)) 6 5 1 3 (10^(0.0)) 11 11 4 (10^(−1.0)) 12 125 (none) 14 14

[0041] As determined by the CST methodology, the protection afforded byin ovo vaccination at incubation day 18 with IB virus/vaccine derivedfrom Poulvace IB MM against exposure to a virulent challenge IB virus at3 weeks of age was excellent. The protection percentages ranged from alow of 92% to a high of 100%.

[0042] In addition to the hatchability, clinical signs, and CST analysesdiscussed above, a serological study was also performed. In brief, bloodsamples were collected from the wing veins of all chicks up to a maximumnumber of 24 chicks per group at 3 weeks of age. Lacrimal fluid wascollected after dropping 1 drop of glycerin into each eye from a maximumnumber of 5 chicks per group at 3 weeks of age. Antibody titers againstthe IB M41 antigen were measured in serum and lacrimal fluid using theHI test. The detection limit of the HI test corresponds with ²log HItiter=3.0. Geometric mean titers (hereinafter, “GMT”) were calculatedbased upon the HI tests conducted. Tables 8 and 9 present theserological results. TABLE 8 Calculated Serological Results GMT againstIB M41 antigen at 3 weeks of age using the HI test serum lacrimal fluidGroup (EID₅₀) (# chicks) (# chicks) 1 (10^(2.0)) 3.0 (4) 8.3 (3) 2(10^(1.0)) 3.0 (6) 7.5 (2) 3 (10^(0.0)) 3.1 (11) 7.4 (5) 4 (10^(−1.0))3.1 (11) 8.7 (3) 5 (none) 3.0 (14) 9.7 (4)

[0043] TABLE 9 Raw Serological Results # chicks with indicated ²log HItiters against IB M41 antigen at 3 weeks of age Group (EID₅₀) 0 titerresults based upon serum: 1 (10^(2.0)) 2 (10^(1.0)) 3 (10^(0.0)) 0 4(10^(−1.0)) 0 5 (none) 4 titer results based upon lacrimal fluid 1(10^(2.0)) 2 (10^(1.0)) 3 (10^(0.0)) 4 (10^(−1.0)) 5 (none)

[0044] In almost all chicks, antibody levels within the serum were notabove the detection limit of ²log HI titer=3.0. Antibody titers in thelacrimal fluids of both the inoculated chicks and the control chickswere high. Accordingly, it is postulated that: (a) within the lacrimalfluids, the experimentally-determined antibody titers were non-specific;and (b) the glycerin used to collect the lacrimal fluids might have beenresponsible for this observed effect. No clear conclusions were drawnbased upon these serological results discussed above.

[0045] Based upon the entirety of this Example 2 as described above, itwas concluded that in ovo vaccination at day 18 of incubation of SPFchicken eggs with IB virus/vaccine at dosages of ranging from a low of

[0046] 10^(−1.0) EID₅₀ per egg to a high of 10^(2.0) EID₅₀ per egg wasefficacious in protecting chicks against exposure to a challenge at 3weeks of age with virulent IB M41 virus.

D) EXAMPLE 3 Efficacy Study for in Ovo Vaccination of Commercial ChickenEggs

[0047] Commercial chicken eggs for broilers were obtained from Pronk,Meppel, The Netherlands. These eggs were incubated within appropriatefacilities. After 18 days incubation, all eggs were candled, and 4groups of 28-30 eggs were inoculated with graded doses of the in ovovaccine. The in ovo vaccines administered, hatching and/or husbandryconditions, the manner of egg injections, the preparation of thechallenge virus, serological analysis, and the determination ofprotection using the CST methodology were all conducted as previouslydescribed above in Examples 1 and 2. Tables 10-16 below present theresults from this study of in ovo vaccination with IB virus ofcommercial chicken eggs. TABLE 10 Calculated Hatchability Resultshatchability results after in ovo vaccination at incubation day 18 witha dose of 0.05 ml/egg of IB vaccine to Groups 1-5 (28-30 eggs/group) #hatched Group EID₅₀ (total # eggs) % hatched 1 10^(2.0) 25 (29) 86 210^(1.0) 26 (28) 93 3 10^(0.0) 25 (28) 89 4  10^(−1.0) 27 (30) 90 5 none24 (28) 86

[0048] TABLE 11 Raw Hatchability Results hatchability results after inovo vaccination at incubation day 18 with a dose of 0.05 ml/egg of IBvaccine to Groups 1-5 (28-30 eggs/group) # hatched # hatched # hatchedtotal # Group EID₅₀ (day 20) (day 21) (day 22) hatched 1 10^(2.0) — 22 3 25 2 10^(1.0)  2 24 — 26 3 10^(0.0) 18  7 — 25 4  10^(−1.0) 18  9 —27 5 none 13 11 — 24

[0049] As set forth above in Tables 10 and 11, hatchability in theinoculated groups (i.e., Groups 1-4) was good, within customary limits,and ranged from 86% to 93%. Hatchability in the control group (i.e.,Group 5) was 86%. TABLE 12 Clinical Signs In Non-Challenged Chickspost-hatch (PH) mortality prior to challenge # days Group 1 Group 2Group 3 Group 4 Group 5 PH (n = 25) (n = 26) (n = 25) (n = 27) (n = 24)1  1 died  1 BC 5  1 died (yolk sac) 7  2 BC 9  1 died  1 killed # live23 26 25 27 22 chicks (day 21 PH)

[0050] Table 12 above sets forth the conditions and mortality of thechicks from Groups 1-5 prior to challenge with a virulent strain of IBvirus. In Table 5, “BC” is used as an abbreviation for bad condition. Tosummarize the results: 2 chicks in Group 1 died (1 from yolk sacinflammation); and 2 control chicks from Group 5 were in bad condition(1 died on day 9 post-hatch and the other 1 chick was killed on day 9post-hatch because it could not stand upright). Table 12 does notpresent clinical respiratory signs. Several chicks in groups givendosages of 100.0 or greater (i.e., Groups 1-3) showed mild respiratorysigns probably due to IB virus replication from 6 days of age onwarduntil challenge at 3 weeks of age (Groups 1 and 2) or until 12 days ofage (Group 3). The observed respiratory signs were mild and no damagewas seen at 6 days post-challenge as determined by the CST methodology(presented below).

[0051] Tables 13 and 14 presents the results of the challenge study.TABLE 13 Calculated Results Of Post-challenge Protection Protectionagainst challenge at 3 weeks of age with virulent IB virus as determinedby CST Group (EID₅₀) # chicks protection % 1 (10^(2.0)) 23 100 2(10^(1.0)) 25 100 3 (10^(0.0)) 25 96 4 (10^(−1.0)) 27 89 5 (none) 22 0

[0052] TABLE 14 Raw Results Of Post-Challenge Protection post-challengeassessments of protection with CST techniques Group +(full CST±(impaired −(no CST (EID₅₀) # chicks motion) CST motion) motion 1(10^(2.0)) 23 23 0 0 2 (10^(1.0)) 26 25 0 0 3 (10^(0.0)) 25 23 2 0 4(10^(−1.0)) 27 23 2 2 5 (none) 22 0 0 22

[0053] In Tables 13 and 14, 1 chick in Group 2 was in bad condition fromthe day after challenge until its death at 5 days post-challenge. Thisdeath was not attributed to either the in ovo vaccination or thesubsequent challenge. As determined by the CST methodology, theprotection afforded commercial eggs for broilers by in ovo vaccinationat incubation day 18 with IB virus/vaccine derived from Poulvac® IB MMagainst exposure to a virulent challenge IB virus at 3 weeks of age wasexcellent. The protection percentages ranged from a low of 89% to a highof 100%. The control chicks (Group 5) had no protection againstchallenge with a virulent IB virus at 3 weeks of age.

[0054] Serological analysis yielded the results set forth below inTables 15 and 16. TABLE 15 Calculated Serological Results GMT against IBM41 antigen at 3 weeks of Group age using the HI (EID₅₀) test of serumsamples # chicks 1 (10^(2.0)) 4.5 23 2 (10^(1.0)) 4.0 22 3 (10^(0.0))4.1 24 4 (10^(−1.0)) 4.0 24 5 (none) 3.8 22

[0055] TABLE 16 Raw Serological Results # chicks with indicated ²log HItiters to IB M41 antigen at 3 weeks of age as determined from serumGroup (EID₅₀) 3 4 5 6 7 1 (10^(2.0)) 8 2 9 2 2 2 (10^(1.0)) 11 3 5 3 3(10^(0.0)) 8 7 7 2 4 (10^(−1.0)) 9 7 7 1 5 (none) 11 5 5 1

[0056] As set forth above in Tables 15 and 16, serological analysis ofserum samples revealed mean antibody titers in all inoculated groupswere only slightly higher than those of the control chicks (i.e., Group5). However, within the inoculated groups, the highest mean antibodytiter was measured in Group 1 chicks; i.e., the chicks which hadreceived the strongest dose of the in ovo vaccines. Additionally, in thevaccinated chicks, a considerable number showed antibody titers at orabove the detection level.

[0057] Based upon the entirety of this Example 3 as described above, itwas concluded that in ovo vaccination at day 18 of incubation ofcommercial chicken eggs for broilers with IB virus/vaccine at dosages ofranging from a low of 10^(−1.0) EID₅₀ per egg to a high of 10^(2.0)EID₅₀ per egg was efficacious in protecting chicks against exposure to achallenge at 3 weeks of age with virulent IB M41 virus.

[0058] Although the present invention has been described above inconsiderable detail, applicants desire the full extent of patentprotection possible as defined and determined by the claims appendedhereto, with reference to the above teachings but not limited to anyspecific example previously set forth.

What is claimed is:
 1. An in ovo vaccine for protecting chickens andother poultry having maternal antibodies to infectious bronchitis (IB)virus from exposure to virulent IB virus, wherein said vaccine comprisesa solution containing, on a chicken egg basis, a live avirulent strainof infectious bronchitis virus in an immunogenically-effective amountwithin the range of about 10^(−1.0) EID₅₀ per egg to about 10^(2.0)EID₅₀ per egg, wherein said vaccine has a percentage (%) protection inpost-hatch member chicks surviving at 3 weeks of age of at least 89%against challenge from virulent IB virus.
 2. The vaccine of claim 1,wherein the immunogenically-effective amount does not decrease thepercentage of in ovo vaccinated chicken eggs that hatch upon theexpiration of the incubation period below 72%.
 3. A poultry vaccineagainst infectious bronchitis virus (IBV) comprising a live avirulentstrain of infectious bronchitis virus in an immunologically effectiveamount for in ovo administration of about 10^(−1.0) EID₅₀ per dose toabout 10^(2.0) EID₅₀ per dose, wherein said vaccine contains infectiousbronchitis virus strain 1263 of the Massachusetts serotype.
 4. A methodof vaccinating a poultry animal against infectious bronchitis (IB),which comprises obtaining a commercial vaccine against IB andadministering said vaccine in ovo to a member selected from the groupconsisting of chickens, turkeys, ducks, geese, bantams, quail andpigeons, said member having maternal antibodies to IB virus, whereinsaid vaccine contains a live, avirulent strain of IB virus (IBV) in aquantity sufficient to confer immunity in an amount within the range ofabout 10^(−1.0) EID₅₀ per dose to about 10^(2.0) EID₅₀ per dose, andfurther wherein said method results in a percentage (%) protection inpost-hatch member chicks surviving at 3 weeks of age of at least 89%against challenge from virulent IBV.
 5. The method of claim 4, whereinsaid vaccine contains about 10^(0.0) EID₅₀ per dose to about 10^(2.0)EID₅₀ per dose.
 6. The method of claim 5, wherein said vaccine containsabout 1.0^(0.0) EID₅₀ per dose to about 10^(1.0) EID₅₀ per dose.
 7. Themethod of claim 4, wherein said vaccine is reconstituted prior toadministration.
 8. The method of claim 4, wherein said vaccine has notbeen approved or indicated for in ovo administration.
 9. The method ofclaim 4, wherein said vaccine contains infectious bronchitis virusstrain 1263 of the Massachusetts serotype.
 10. The vaccine of claim 1,wherein the vaccine contains substantially no virus neutralizing factor.11. The vaccine of claim 3, wherein said vaccine comprises about10^(0.0) EID₅₀ per dose to about 10^(2.0) EID₅₀ per dose.
 12. Thevaccine of claim 11, wherein said vaccine comprises about 10^(0.0) EID₅₀to about 10^(1.0) EID₅₀ per dose.